Bottom Line:
In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3.Increased numbers of macrophages were found on days 1, 3, 7 and 90.Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.

Background: Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.

Methods: Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage fluid (BALf) were determined. Infiltration of granulocytes, macrophages and lymphocyte subsets (CD45RA+, CD5+CD4+, CD5+CD8+) was measured by flowcytometry in BALf, lung parenchyma, thoracic lymph nodes and spleen. Histological analysis was performed on HE sections.

Results: LIRI resulted in hypoxemia, impaired left lung compliance, increased capillary permeability, surfactant conversion, and an increase in MMP-2 and MMP-9. In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3. Increased numbers of macrophages were found on days 1, 3, 7 and 90. Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.

Conclusion: The short-, and long-term changes after LIRI in this model are similar to the changes found in both PAGF and ARDS after clinical lung transplantation. LIRI seems an independent risk factor for the development of PAGF and resulted in progressive deterioration of lung function and architecture, leading to extensive immunopathological and functional abnormalities up to 3 months after reperfusion.

Figure 8: Histological examples of atelectasis (10*) on HE slides. Severe atelectasis was demonstrated up to day 90 after LIRI. HE = Haematoxylin and Eosin staining; LIRI = Lung Ischemia-Reperfusion Injury.

Mentions:
LIRI resulted in diffuse alveolar damage consisting of severe intra-alveolar edema up to day 3, septal edema, which was mild on day 1 and increased to moderate on day 3, and intra-alveolar hemorrhages (Figure 7 this manuscript; see additional file 1, Table 9). The overall classification of LIRI animals changed from exsudative on day 1 to proliferative from day 3 to day 90. Although no atelectasis and fibrosis were seen on day 1 following LIRI, mild fibrosis and mild to severe atelectasis were seen from day 3 up to day 90 after LIRI (Figure 8 this manuscript; see additional file 1, Table 9). Identification of infiltrating cells confirmed the flowcytometry measurements. A mild inflammatory pattern consisting of histiocytes was found on day 3 and 7 in sham-operated animals. LIRI caused moderate to severe inflammation, which changed from mixed (granulocytic, lymphocytic, and histiocytic) inflammation on day 1 to a histiocytic and lymphocytic pattern from day 3 to 90 (Figure 9 this manuscript; see additional file 1, Table 10). No major differences between unoperated, sham-operated and LIRI animals were found in the right lung (data not shown).

Figure 8: Histological examples of atelectasis (10*) on HE slides. Severe atelectasis was demonstrated up to day 90 after LIRI. HE = Haematoxylin and Eosin staining; LIRI = Lung Ischemia-Reperfusion Injury.

Mentions:
LIRI resulted in diffuse alveolar damage consisting of severe intra-alveolar edema up to day 3, septal edema, which was mild on day 1 and increased to moderate on day 3, and intra-alveolar hemorrhages (Figure 7 this manuscript; see additional file 1, Table 9). The overall classification of LIRI animals changed from exsudative on day 1 to proliferative from day 3 to day 90. Although no atelectasis and fibrosis were seen on day 1 following LIRI, mild fibrosis and mild to severe atelectasis were seen from day 3 up to day 90 after LIRI (Figure 8 this manuscript; see additional file 1, Table 9). Identification of infiltrating cells confirmed the flowcytometry measurements. A mild inflammatory pattern consisting of histiocytes was found on day 3 and 7 in sham-operated animals. LIRI caused moderate to severe inflammation, which changed from mixed (granulocytic, lymphocytic, and histiocytic) inflammation on day 1 to a histiocytic and lymphocytic pattern from day 3 to 90 (Figure 9 this manuscript; see additional file 1, Table 10). No major differences between unoperated, sham-operated and LIRI animals were found in the right lung (data not shown).

Bottom Line:
In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3.Increased numbers of macrophages were found on days 1, 3, 7 and 90.Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.

Background: Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.

Methods: Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage fluid (BALf) were determined. Infiltration of granulocytes, macrophages and lymphocyte subsets (CD45RA+, CD5+CD4+, CD5+CD8+) was measured by flowcytometry in BALf, lung parenchyma, thoracic lymph nodes and spleen. Histological analysis was performed on HE sections.

Results: LIRI resulted in hypoxemia, impaired left lung compliance, increased capillary permeability, surfactant conversion, and an increase in MMP-2 and MMP-9. In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3. Increased numbers of macrophages were found on days 1, 3, 7 and 90. Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.

Conclusion: The short-, and long-term changes after LIRI in this model are similar to the changes found in both PAGF and ARDS after clinical lung transplantation. LIRI seems an independent risk factor for the development of PAGF and resulted in progressive deterioration of lung function and architecture, leading to extensive immunopathological and functional abnormalities up to 3 months after reperfusion.